Cells must be able to quickly respond to changes in their environment. An important aspect of many such responses is the fast and reversible modification of proteins to regulate their function. The transient phosphorylation of proteins through the combined action of kinases and phosphatases may be the most prominent example of such regulation. A similar, more recently discovered modification of proteins is the stabile addition of adenosine 5'- monophosphate group (AMP) to proteins. This novel regulatory mechanism has been called AMPylation after its initial discovery in the context of the bacterial VopS protein. Since then, bacterial and eukaryotic Fic domains have been found that can AMPylate proteins. Drosophila offers a significant advantage for the analysis of the physiological role of AMPylation because fly genomes encode only a single Fic domain protein. The specific aims described in this proposal combine biochemical and genetic approaches in Drosophila to define the role that this mechanism plays in visual neurotransmission. This proposal aims (i) to determine the cellular sites of AMPylation activity using immunofluorescence and electron microscopy approaches, (ii) to define developmental stages and cell types that require AMPylation, (iii) to analyze the physiological consequences of AMPylation in the context of the Drosophila visual system, and (iv) to employ biochemical purification in conjunction with genetic interaction assays to identify the molecular targets regulated by AMPylation. Completion of these experiments will yield a comprehensive understanding of the signaling pathways in the visual system that is modified by AMPylation. PUBLIC HEALTH RELEVANCE: AMPylation is the stabile addition of an adenosine 5'-monophosphate group (AMP) to proteins. Recent findings have revealed that this novel regulatory mechanism plays an important role in diverse physiological process in eukaryotic cells. This proposal combines biochemical and genetic approaches in Drosophila to define the role that this novel mechanism plays in visual neurotransmission. Understanding the mechanism by which AMPylation modifies photoreceptor responses is an important first step so that we can start to appreciate the potential of this novel layer of regulation in modulating visual processing in health and disease.